肝星状细胞SCAP特异性敲除对小鼠肝纤维化的影响  

Effects of hepatic stellate cell-specific deletion of SCAP on hepatic fibrosis in mice

作　　者：侯晓俐 饶雨涵 姚盈程 苏玉 李丹阳 陈压西 HOU Xiao-Li;RAO Yu-han;YAO Ying-cheng;SU Yu;LI Dan-yang;CHEN Ya-xi(Key Laboratory of Metabolism on Lipid and Glucose,Center for Lipid Research,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学脂糖代谢性疾病重庆市重点实验室,脂质研究中心,重庆400016

出　　处：《中国病理生理杂志》2022年第2期250-258,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82170586;No.81900406)。

摘　　要：目的:探讨固醇调节元件结合蛋白裂解活化蛋白(SCAP)在肝星状细胞中缺失能否延缓小鼠肝纤维化进程。方法:将SCAP^(loxP/loxP)小鼠与Lrat-Cre工具鼠繁殖得到Lrat-Cre^(+/+)SCAP^(fl/fl)、Lrat-Cre^(+/−)SCAP^(fl/fl)和Lrat-Cre^(−/−)SCAP^(fl/fl)小鼠,并用PCR法对小鼠基因型进行鉴定。选取4~6周龄Lrat-Cre^(−/−)SCAP^(fl/fl)小鼠(雄性,n=8)作为对照(Con)组,Lrat-Cre+/+SCAP^(fl/fl)和Lrat-Cre^(+/−)SCAP^(fl/fl)小鼠(雄性,n=8)作为实验(SCAP^(−/−))组,均给予高脂饮食16周。称量小鼠肝重和体重,计算肝指数;检测血清总胆固醇(TC)、甘油三酯(TG)、丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)水平;苏木精-伊红(HE)染色观察肝脏形态,油红O染色观察肝脏脂质积聚,天狼星红染色评估肝脏纤维化程度;RT-qPCR和Western blot检测小鼠肝组织中TGF-β/Smad信号通路相关mRNA及蛋白表达。结果:小鼠基因型鉴定结果与预期一致。提取两组小鼠原代肝星状细胞,RT-qPCR结果显示SCAP^(−/−)小鼠肝星状细胞中SCAP mRNA表达显著降低(P<0.01),免疫荧光染色显示SCAP^(−/−)小鼠肝星状细胞中SCAP不表达,表明SCAP^(−/−)小鼠模型建立成功。与Con组相比,高脂饮食喂养的SCAP^(−/−)组小鼠体重(P<0.01)和肝重(P<0.05)显著降低,但肝指数无显著差异;血清中TC水平显著降低(P<0.05),TG、AST和ALT水平无显著差异;HE及油红O染色结果显示小鼠肝脏脂肪变性和脂质积聚无显著差异;天狼星红染色显示肝脏纤维化程度减轻(P<0.05);Smad2(P<0.05)和Smad3(P<0.01)的mRNA表达显著降低;Smad2/3和p-Smad2/3蛋白水平显著降低(P<0.05)。结论:肝星状细胞中SCAP缺失可延缓小鼠肝纤维化发展的进程,并与TGF-β/Smad信号通路有关。AIM:To investigate whether sterol regulatory element-binding protein cleavage-activating protein(SCAP)deletion in hepatic stellate cells attenuates the progression of liver fibrosis in mice.METHODS:The SCAPloxP/loxP mice were bred with Lrat-Cre tool mice to obtain Lrat-Cre+/+SCAPfl/fl,Lrat-Cre+/−SCAPfl/fl and Lrat-Cre−/−SCAPfl/fl mice,and the genotypes of the mice were identified by PCR.The Lrat-Cre−/−SCAPfl/fl mice aged 4 to 6 weeks(male,n=8)were selected as control(Con)group,while the Lrat-Cre+/+SCAPfl/fl and Lrat-Cre+/−SCAPfl/fl mice(male,n=8)served as experimental(SCAP−/−)group.The mice in both groups were fed with high-fat diet for 16 weeks.The liver weight and body weight of the mice were measured,and the liver index was calculated.The serum was collected,and the levels of total cholesterol(TC),triglycerides(TG),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were recorded.Hema⁃toxylin-eosin(HE)staining was used to observe the morphological changes of liver tissues,oil red O staining was used to observe the lipid accumulation,and Sirius red staining was used to evaluate the fibrosis of the liver.The TGF-β/Smad mRNA and protein levels in liver tissues were determined by RT-qPCR and Western blot,respectively.RESULTS:The results of genotype identification were the same as expected.The results of RT-qPCR showed that the mRNA expression of SCAP in primary hepatic stellate cells extracted from SCAP−/−mice was significantly decreased(P<0.01),and the immu⁃nofluorescence staining results showed that SCAP was not expressed in the hepatic stellate cells from SCAP−/−mice,indi⁃cating that the hepatic stellate cell-specific SCAP−/−mouse model was successfully established.Compared with Con group,the body weight(P<0.01)and liver weight(P<0.05)of SCAP−/−mice fed with high-fat diet decreased,but there was no significant change in liver index.The level of TC in serum was decreased(P<0.05),while TG,AST and ALT had no sig⁃nificant difference.HE staining and oil red O stai

关 键 词：固醇调节元件结合蛋白裂解活化蛋白 肝星状细胞 肝纤维化 TGF-Β/SMAD信号通路 

分 类 号：R575.5[医药卫生—消化系统] R363.2[医药卫生—内科学]